Combinatorial chemistry, a new approach to the identification and optimization of drug leads in medicinal chemistry, has been enormously successful in synthesizing large number of compounds for pharmacological screening and testing (Gordon et al., 19%; Thompson and Ellman, 1996). In response to the large number of new compounds produced, high throughput screening methods are being developed to rapidly identify lead compounds in combinatorial libraries that interact with specific receptors, or show desirable pharmacological effects in bioassays (Loo, 1997). As the number of lead compounds being identified through combinatorial methods increases substantially, preclinical investigations such as the investigation of drug metabolism and toxicity become the new bottleneck to the process of bringing new drugs to market.
Traditionally, live animals or perfused liver or gut preparations have been used during the preclinical investigation of metabolism and toxicity of drugs and other xenobiotic compounds. In an effort to reduce costs, to reduce the number of animals used, and to gain insights into toxicological pathways, in vitro assays have been developed to study drug metabolism using hepatic microsomes, reconstituted purified isozymes, primary culture hepatocytes, tissue slices, and cytochrome P450 overexpressed in whole cells (Parkinson, 1996; Maurel, 1996). However, these in vitro assays lack sufficient throughput to keep pace with the large number of lead compounds being identified through combinatorial chemistry drug discovery programs. The fastest current method to assess drug metabolism utilizes hepatic microsomes, which are incubated in a test tube with a drug and the cofactor NADPH. After at least 10 minutes, the solution is extracted (5-30 min), the extract may be concentrated (0-60 min), then the extract is analyzed using HPLC, GC, LC-MS or GC-MS (10-60 min). Therefore, the fastest current method requires from 35-170 min, depending upon the sample handling and analysis procedures. There is a need to streamline (reduce the number of separate steps, reduce the sample handling requirements, and integrate the process into a faster, simpler and automated assay) and increase the throughput of existing in vitro metabolism assays. In addition, no method exists to readily measure the intrinsic bioavailability of a compound, in particular on a large scale, high throughput basis. Pulsed ultrafiltration mass spectrometry was applied to metabolic screening of xenobiotic compounds by the inventors (Van Breemen et al. 1998).